Sulpholipid composition and methods for treating skin disorders

ABSTRACT

The invention refers to the use of a sulpholipid SQDG having formula (I), wherein R 1  and R 2 , which are the same or different, are hydrogen or saturated or unsaturated, optionally hydroxy-substituted acyl groups, protonated or as a salt, for the preparation of a pharmaceutical composition for the treatment of inflammatory skin diseases or disorders, especially psoriasis. A pharmaceutical composition comprising an SQDG compound in combination with a polyunsaturated fatty acid and a therapeutically acceptable carrier is also described. ##STR1##

The present application is a U.S. National application filed under 35USC 371 of PCT/SE97/00735, filed Apr. 20, 1997.

A SULPHOLIPID COMPOSITION AND METHODS FOR TREATING SKIN DISORDERS

The present invention refers to a new PAP-receptor antagonist, thesulpholipid SQDG, as well as to the use of SQDG for the prophylaxis ortreatment of inflammatory skin diseases, especially psoriasis.

BACKGROUND OF THE INVENTION

Inflammatory skin diseases such as atopic dermatitis, urticaria andespecially psoriasis still constitute a great problem for the affectedpatients as there are today no effective therapies.

Psoriasis is a common chronic inflammatory dermatosis with a globaldistribution; it has been estimated that about 1.5% of the population inthe western countries can be expected to suffer from the disease duringtheir lifetime. A number of different clinical patterns of psoriasis areacknowledged, the most common being plaque psoriasis.

Hyperproliferation, inflammation with massive infiltration of leucocytesand disturbances in cell differentiation are the typical characteristicsof psoriatic skin. The number of basal cells is vastly increased whichreduces the turn-over time for the epidermis from the normal 27 days to3-4 days. The normal events of cell maturation and keratinization do notoccur. This proliferation of keratinocytes occurs both in involved andnon-involved psoriatic skin but is most pronounced in the plaques. Theinflammatory infiltrate from psoriatic lesions has been found to consistpredominantly of mononuclear T lymphocytes. There is a disturbed T cellfunction also in the circulating blood, which implies a possiblecell-mediated immunological abnormality in psoriasis. The phagocytesthat can be identified histologically in the psoriatic lesion,neutrophils in particular, suggest a role for the chemotacticinflammatory mediators in the pathology of psoriasis. Among theseinterleukin-1, -6 and -8, leukotriene B₄ and PAF have been isolated inpathological amounts in the affected skin.

There is a number of different therapies of psoriasis of varyingeffectiveness, none being perfect. Among the antipsoriatic drugs ofnatural origin can be mentioned coal tar, dithranol, psoralens,retinoids and cyclosporin A. In addition to said more establishedtherapies can also be mentioned the use of podophyllotoxin, a lignanwhich can be isolated from Podophyllum species, polyunsaturated fattyacids, such as eicosapentaenoic acid and gammalinolenic acid, andcolchicine, an alkaloid from the crocus plant. The use of essentialfatty acids in atopic dermatitis and psoriasis, respectively, aredescribed by Wright, S., British Journal of Dermatology 125, 503-515,1991.

In the traditional medicine of Honduras the name Calaguala is used forthe extract of a number of closely related Polypodium species, includingP. decumanum Willd., P. aureum (or P. leucatomos), P. lowei C. Chr., P.loriceum L., P. triseriale Sw., P. fraxinifolium Jacq. and P. dissimileL (Molina, personal communication, 1991). A decoction or infusion of theCalaguala plant has been used to treat a number of diseases includingpeptic ulcer, kidney problems, diarrhoea and arthritis or other pains injoints and tendons.

Over the last two decades several clinical trials have been performed oncalaguala in the treatment of psoriasis, as well as atopic dermatitis(Vargas Gonzales, J. P., et al., Acta Pediatric. Esp. 46(1), 556-561,1988) and vitiligo (Gonzales, S., et al., J. Invest. Derm. 102(4), 651,1994).

Platelet activating factor (PAF;1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) is a phospholipidderived mediator with a diversity of biological effects. PAF is releasedfrom various cell types, including platelets, neutrophils, monocytes andendothelial cells. In addition, several cell types, includingneutrophils, are known to express specific PAF receptors on their cellmembrane. In vitro PAF exhibits effects, including aggregation anddegranulation of leukocytes and inhibition of lymphocyte proliferation.In vivo effects include hypotension, acute renal failure and increase invascular permeability. PAF is thought to be associated with a number ofpathological conditions, such as shock, airway hypersensitivity andasthma, inflammation of many different types, psoriasis, arthritis andgraft rejection and various cardiovascular disorders especially onesassociated with thrombosis, blood coagulation and platelet aggregation.

A large number of natural and synthetic PAF receptor antagonists hasbeen discovered which show different physiological effects, but to datethey have been of limited usefulness for pharmaceutical purposes. Theuse of some compounds in the treatment of asthma is, however, currentlybeing evaluated. As the PAF antagonist therapy is relatively new, moreresearch is still needed for the judgement of therapeutic benefit forspecific indications. It is believed that safe and potent compounds willhave a substantial role in therapeutical treatments.

Perhaps the best known of all PAF antagonists is the ginkgolide BN 52021which was isolated from the "fossil tree" Ginkgo biloba in the late60's. It is a specific PAF receptor antagonist and has shownanti-inflammatory properties in several in vivo and in vitro models. Forthe potential oral treatment of chronic inflammatory diseases, thesynthetic hetrazepine derivative BN 50730 was later developed. It showsa several ten-folds more potent PAF antagonistic activity in vitro.(Guinot, P. in Clinical Reviews in Allergy 12, 397-417, 1994.)

It has been suggested that PAF contributes to the inflammatory aspectsof psoriasis (Cunningham, 1990). Factors that implicate its role in thepathogenesis and symptoms are that PAF has been isolated in elevatedamounts on psoriatic skin, that injection of PAF causes vasodilation andincreased vascular permeability, and that PAF is chemoattractant mainlyto neutrophils.

Recently, PAF activity has been measured in blood plasma of patientswith psoriasis using a radioimmunoassay technique (Izaki et al., BritishJournal of Dermatology 136, 1060-1064, 1996). It was shown that levelsof PAF were significantly elevated in patients with psoriasis and thatwhen these patients were treated with conventional anti-psoriaticmethods the levels were decreased. Also, the decrease was correlatedwith a clinical improvement. It is concluded that PAF has a role in theacute phase of psoriatic and leucotactic inflammation.

In another study, chemotactic responsiveness of pheripheral bloodeosinophils from healthy subjects and from patients with inflammatorydermatoses (psoriasis, atopic dermatitis) was determined. It was foundthat chemotactic responsiveness was significantly enhanced in severelyaffected patients and that this increase was not related to a specificdisease. The increased responsiveness in peripheral eosinophils to PAFis suggested to be related to altered receptor expression duringcutaneous inflammation (Morita et al. 1989).

PRIOR ART

The sulphoquinovosyldiacylglycerols, SQDG, constitute one of the fourmajor classes of polar lipids in chloroplast membranes of mostphotosynthetic organisms. They are structural components of thethylakoid membrane and are involved in the electron transport chain inphotosynthesis as well as in the construction of chloroplasts.

In Chem. Pharm. Bull. 41 (9), 1664-1666, 1993, Shirahashi et al.describe the isolation and identification of anti-tumour-promotingprinciples from an extract of the cyanobacterium Phormidium tenue. Saidextract turned out to comprise three classes of glycolipids, that ismonogalactosyldiacylglycerol (MGDG), digalactosyldiacylglycerol (DGDG)and sulphoquinovosyldiacylglycerol (SQDG). MGDG and DGDG turned out toinhibit tumour promotion more intensely than SQDG, or rather a mixtureof different SQDGs.

The Journal of the National Cancer Institute, Vol. 81, No. 16, Aug. 16,1989, Gustafson et al., refers to the isolation and identification offour anti-HIV active sulpholipids from cyanobacterial extracts by acombination of gel-permeation and reversed-phase chromatographies. Thesulpholipids all had similar levels of activity.

JP patent application No. 64-186626 discloses the use of asulphonoglycolipid, an SQDG of the formula ##STR2## to obtain a remedyfor intravascular blood coagulation syndrome, and JP patent applicationNo. 64-186627 describes the use of the same compound in the treatment ofnephritis.

DESCRIPTION OF THE INVENTION

The present invention refers to the use of an SQDG having the formula##STR3## wherein R¹ and R², which are the same or different, arehydrogen or saturated or unsaturated, optionally hydroxy-substitutedacyl groups, protonated or as a salt, for the preparation of apharmaceutical composition for the treatment of inflammatory skindiseases or disorders.

A preferred SQDG to be used according to the invention is asulphoquinovosyldiacylglycerol having the formula ##STR4## wherein R¹and R², which are the same or different, are hydrogen, acetyl or acylgroups comprising 14-22 carbon atoms, preferably 16-18, and having up to6 unsaturations, preferably 0-3.

As examples of acyl groups R¹ and R² can be mentioned fatty acidresidues such as palmitoyl, stearoyl, oleoyl, linoleoyl, γ-linolenoyl,columbinoyl, dihomo-γ-linolenoyl, arachidonoyl, eicosapentaenoyl,docosahexaenoyl and ricinoloyl, as well as conjugated forms thereof.

The invention specially refers to the use of a1,2-O,O-dipalmitoyl-[6'-sulpho-α-D-quinovopyranosyl(1'→3)]-sn-glycerol,an SQDG having the formula ##STR5## protonated or as a salt.

Whenever applicable, the expression "an SQDG" is also intended tocomprise a salt of SQDG, for instance a sodium, potassium or ammoniumsalt, or a mixture of one or more SQDG compounds. The counterion canalso be a cationic lipid or another organic cation to form an SQDGderivative being a prodrug. The SQDG compounds may be isolated fromnature as such or may be chemically modified to introduce different acylgroups with specific valuable properties. Examples of modifications arepartial or total hydrolysis of acyl groups, partial or totalhydrogenation, regioselective desaturation and inter- ortransesterification. In natural SQDGs the quinovosyl is always inposition 3 of the glycerol and has the D-form. The glycosidic link ingeneral has the α-form.

Especially, the SQDG can be used according to the invention for theprophylaxis or treatment of atopic dermatitis, urticaria and psoriasis.

Another use of an SQDG according to the invention is as anantiproliferative agent.

A preferred use of an SQDG according to the invention is for theprophylaxis or treatment of psoriasis.

Another preferred use of an SQDG according to the invention, is incombination with a mono- or polyunsaturated fatty acid for theprophylaxis or treatment of psoriasis. In this combination the fattyacids can be in the form of free fatty acids, monoacylglycerols,diacylglycerols or triacylglycerols, for example a marine oil or eveningprimrose oil.

Mono-unsaturated fatty acids preferably comprise 16-22 carbon atoms, andcan for instance be oleic acid.

The polyunsaturated fatty acid, PUFA, to be used in combination with theSQDG should preferably comprise 18-22 carbon atoms and 2-6unsaturations, and can for instance be an essential fatty acid of then-6 or n-3 family. Examples of n-6 acids are γ-linolenic acid,dihomo-γ-linolenic acid, arachidonic acid. Examples of n-3 acids areα-linolenic acid, octadecatetraenoic acid, eicosatetraenoic acid,eicosapentaenoic acid, docosapentaenoic acid and docosahexaenoic acid.

In the treatment of atopic dermatitis the fatty acids are preferablychosen among γ-linolenic acid, dihomo gammalinolenic acid andarachidonic acid. In the treatment of psoriasis the fatty acids arepreferably chosen among α-linolenic acid, octadecatetraenoic acid,eicosatetraenoic acid, eicosapentaenoic acid, docosapentaenoic acid anddocosahexaenoic acid.

The invention also refers to a pharmaceutical composition comprising anSQDG having the formula ##STR6## wherein R¹ and R², which are the sameor different, are hydrogen or saturated or unsaturated, optionallyhydroxy-substituted acyl groups, protonated or as a salt, in combinationwith a polyunsaturated fatty acid and a therapeutically acceptablecarrier for the treatment of dermal diseases, especially psoriasis.

The pharmaceutical composition preferably comprises an SQDG having theformula II or III as described above.

Therapeutically acceptable carriers are for instance conventionalcarriers for solutions, suspensions, liposomes, emulsions, aerosols,topical preparations, powders, granules, tablets, capsules orsuppositories.

Said pharmaceutical composition can be orally, topically, ocularly,nasally, aurally, vaginally, rectally, enterally or parenterallyadministrated.

In oral administration the administered dose could be from 1 to 100mg/d, preferably 2-25, especially 2-15 mg/d. In topical application theadministered dose should be at least ten times the oral dose giving apreferred range of 10-200 mg/cm^(2/) d. The injected amount of theactive substance could be estimated to 0.1 to 10 mg/d.

The invention also refers to a new PAF-receptor antagonist,characterized in being an SQDG having the formula ##STR7## wherein R¹and R², which are the same or different, are hydrogen or saturated orunsaturated, optionally hydroxy-substituted acyl groups, protonated oras a salt.

SQDG can be obtained from natural sources, such as green plants, planttissues, sponges, bacteria or algae by drying, such as air drying orlyophilization, extraction and subsequent purification bychromatographic or other methods. In cases where chemical modificationof the obtained material is performed, further purification may benecessary to obtain the desired compound. In this application SQDG wasobtained by preparing a methanolic extract of the plant material whichwas subsequently subjected to repeated straight phase columnchromatography on silica gel with chloroform and methanol as eluents.Fractions containing polar lipids were then further purified on SephadexLH20 with water, methanol and ethanol mixtures.

DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the dose response curve of the SQDG of the formula III, andof the PAF receptor antagonist ginkgolide BN 52021;

FIG. 2 shows the displacement curves of the ginkgolide BN 52021, the PAFreceptor antagonist BN 50730, and of the SQDG compound of the formulaIII.

Isolation of SQDG From Polypodium Decumanum

The plant material for preparing the Calaguala extract was obtained froma plantation near the Lago de Yohoa in Honduras. Polypodium decumanum isa fern which in its natural environment grows as an epiphyte on palmtrees, but which is also cultured for its beneficial pharmacologicaleffects.

The dried and ground leaves (6.6 kg) were extracted with 3×35 1 methanol(puriss) overnight under stirring at room temperature. The extract wasfiltered, concentrated in vacuo and lyophilised (yield 267 g). 50 g ofthe methanolic extract was then dissolved in 400 ml water and extractedwith 4×600 ml chloroform; yield 16 g. The chloroform extract wasadsorbed on silica gel (Merck, mesh 70-230) and eluted through a columnpacked with 200 g silica gel using a gradient of chloroform to methanol.Fractions of 20 ml were collected and pooled together according to TLCcomparison. 10 fractions were obtained and checked for activity in theelastase assay described below. The most active fractions (1.8 g) wererepeatedly chromatographed on silica gel with a gradient of chloroformto methanol and the combined active fractions (1.4 g) were then elutedthrough a Sephadex LH 20 column (Pharmacia Fine Chemicals, Sweden) witha gradient of methanol (30%) and water to 100% methanol as mobile phase.The next to the last fraction (210 mg) showed the highest activity inthe assay and was further purified using a second Sephadex LH 20 columnwhich was first washed with ethanol and then eluted with methanol. Bythis 10 mg of the compound1,2-O,O-dipalmitoyl-[6'-sulpho-α-D-quinovopyranosyl(1'→3) ]-sn-glycerol,that is a compound of the formula III above, was obtained. The structureof the compound was verified as below.

Identification of the SQDG Compound

The structure of the compound was identified by means of massspectrometry, carbohydrate analysis and NMR.

GC-MS analysis was done on a HP 5890 gas chromatograph coupled to a HP5970A quadruple mass selective detector operated at an ionizationvoltage of 70 eV and an electron multiplier voltage of 1800 V. Thecompound was analyzed after a transesterification reaction according toSeppanen-Laakso, T., et al., Acta Pharm. Fenn. 99, 109-117, 1990, whichallows the identification of bound fatty acids. A single peak wasobserved in the GC chromatogram and the peak exhibited major signals atm/Z (M+) 239, 227, 213, 199, 185, 157, 143, 129, 97, 87, 74, 59, 43 andwas thus identified as the methylester of palmitic acid. For themolecular weight determination a positive ion FAB mass spectrum wasrecorded with a JEOL SX 102 instrument with glycerol as matrix. By thisa molecular ion [M+1]⁺ at m/z=817 was displayed corresponding to amolecular formula of C₄₁ H₇₇ O₁₂ SNa for the compound.

Carbohydrate analysis was performed by hydrolysing the compound withtrifluoroacetic acid followed by reduction and acetylation and theresulting peracetylated compound was compared by gas chromatography withauthentic carbohydrates treated in the same manner. This comparisonyielded glucose.

The NMR experiments were run using a JEOL 270 MHz instrument and amixture of CD₃ OD and CDCl₃ as solvent and TMS as internal standard. The¹ H-NMR and ¹³ C-NMR shift values for the compound are shown in Table 1.The values have been arranged into three groups, that is acyl, glyceroland sulphoquinovosyl moieties.

                  TABLE 1                                                         ______________________________________                                        NMR-values                                                                                .sup.1 H shift                                                                            .sup.13 C shift                                       ______________________________________                                        Acyl moieties                                                                 C16           0.88(t)       14.3                                              C15                         23.4                                              C14                         32.7                                              C methylene   1.25(m)       29.9, 30.2, 30.5                                  C3            1.59(m)       25.7                                              C2            2.3(b)        34.8, 35.0                                        C1                          173.7                                             Glycerol moiety                                                               sn-1*         4.49(dd), 4.17(dd)                                                                          64.0                                              sn-2          5.3(m)        71.3                                              sn-3          4.08(dd), 3.57(dd)                                                                          66.7                                              Sulphoquinovosyl moiety                                                       C1'           4.81(d)       99.0                                              C2'           3.40(dd)      73.0                                              C3'           3.62(t)       74.6                                              C4'           3.10(t)       74.6                                              C5'           4.05(m)       69.4                                              C6'           3.34(dd), 2.94(dd)                                                                          54.0                                              ______________________________________                                         *sn refers to stereospecific numbering                                   

Formulations

Oral Emulsion

An oil-in-water emulsion (batch size 250 g) was prepared with thefollowing ingredients:

    ______________________________________                                        Ingredients            %                                                      ______________________________________                                        Corn oil               20.00                                                  Galactolipid           1.70                                                   SQDG                   0.15                                                   Sucrose                17.00                                                  Potassium sorbate      0.20                                                   Ascorbyl palmitate     0.02                                                   Ammonium phosphatides  0.10                                                   Lemon flavour          0.20                                                   Water                  ad 100.00                                              ______________________________________                                    

The emulsifier (CPL®-Galactolipid, a lipid emulsifier manufactured byScotia LipidTeknik AB, Sweden) and antioxidant were dispersed in theoil. Sucrose, preservative, flavour and water were mixed. The oil phaseand the aqueous phase were preheated to 50° C. and then the oil wasadded to the aqueous phase under high shear mixing at 16,000 rpm for 3.5min. The preemulsion was then homogenised at 80 MPa and 55° C. for 7cycles (Mini-Lab 8.30 H; APV Rannie AS, Denmark). This formulationresulted in a milky emulsion. The daily dose of this emulsion is 10 ml.

The neutral vegetable corn oil can be replaced by for instance an oilrich in γ-linolenic acid, such as evening primrose oil, EPO, giving anemulsion comprising SQDG and PUFA.

    ______________________________________                                        Topical formulation                                                           Ingredient            %                                                       ______________________________________                                        Galactolipid          17.00                                                   SQDG                  2.00                                                    Metagin               0.05                                                    Propagin              0.01                                                    Water                 ad 100.00                                               ______________________________________                                    

SQDG was dispersed in water containing the preservatives. After theaddition of the galactolipids, prepared from oats, the mixture wasalternatively vortexed and stirred giving a viscous dispersion apted fortopical application.

Biological Tests

In vivo animal models to test for anti-psoriatic activity are few andinadequate. Therefore, models that focus on the differentcharacteristics of the disease are frequently used. One possibility isto consider the reported immunological background of the disease and thefact that the immunosuppressive drug cyclosporin A shows excellenttherapeutic effects in treatment of severe psoriasis to use a test modelwhere the ability of a compound to suppress the immune system isstudied. In Phytother. Res. 5, 234-236, 1991, Tuominen et al. report anenhancing effect of Calaguala on the prevention of rejection of skintransplants in mice.

The ability of Calaguala methanolic extract to retard rejection of skingrafts in mice was studied using an adaptation of a method described byBillingham et al., J. Exp. Biol. 28, 385-403, 1957. Mice werepre-treated with either Calaguala orally or subcutaneously or withcyclosporin A subcutaneously for two weeks whereafter they were graftedwith skin from a donor mice of different strain. They were then examineddaily for signs of rejection. The results show that the Calagualamethanolic extract administered in a dose of 500 mg/kg/day gave asignificant enhancement of the rejection time as compared to untreatedanimals. The mechanism for this effect could be the same as for theclinical effects reported for treatment of psoriasis with Calaguala.

The inflammatory aspect of psoriasis can be studied by using arachidonicacid induced oedema in rat ear, Bosman, Skin. Pharmacol. 7, 324-334,1994, and by using the rat paw oedema test, a classical way ofexpertimentally investigating the effect of different compounds in acuteinflammation.

The inflammatory tests described below were performed with themethanolic extract obtained from Polypodium decumanum, as describedabove, the Calaguala extract, in order to evaluate an optional activityof the extract on psoriasis.

It has previously been shown that the Calaguala extract exhibitsdose-dependent inhibition of leukotriene B₄ synthesis in humanleucocytes. This inhibitory activity was found to be caused by thepolyunsaturated fatty acids linoleic, linolenic and arachidonic acid.The activity of additional unsaturated fatty acids, such as oleic acidand eicosapentaenoic acid, was also examined in the model and were allfound to exhibit comparable inhibitory patterns. Leukotriene B₄ is aninflammatory mediator isolated in abnormally high quantities in thepsoriatic skin. See Vasange et al., Prostaglandins, Leukotrienes andEssential Fatty Acids 50, 279-284, 1994.

Rat Ear Oedema

The rat ear oedema test is aimed to be used for detection of drugs withtopical anti-inflammatory activity. It is based on the work by Tonelliet al., Endocrinology 77, 625-634, 1965, but instead of using thecarcinogenic croton oil as induced, ethyl phenyl propiolate (EPP) isapplied topically on the rat ears. The oedema develops rapidly (about 30minutes) and starts to decline after two hours. Several measurements canbe made by using a special measuring device and the values obtained withapplication of both EPP and the drug to be investigated are compared tovalues obtained with EPP alone.

The mechanism of the EPP induced oedema is not completely understood butit is known that compounds with broad anti-inflammatory action, likesteroidal anti-inflammatory drugs, are active in this assay. The oedemacan also be induced by other agents, such as arachidonic acid, and inthat specific case the oedema is believed to develop through the actionof the phospholipid, inflammatory mediators, leukotrienes. They can alsocontribute to the EPP oedema.

The Calaguala methanolic extract was tested in this model on severaloccasions. It was applied either locally (dissolved in methanol oracetone) or orally. Representative experiments are presented below:

                  TABLE 2                                                         ______________________________________                                        Ear thickness                                                                                              Mean decrease                                    Treatment    Number of ears treated                                                                        in oedema                                        ______________________________________                                        None         6               0                                                Calaguala 400 μg/ear                                                                    12              30%                                              Mepyramin    6               44%                                              ______________________________________                                    

The thickness of ears is measured after 2 hours. As positive control theantihistamine mepyramine (1 μg/ear) is used. The significance of thevalues obtained was determined by using Students t-test (one tailedanalysis). For mepyramin the significance was p<0.01and for Calagualap<0.001.

                  TABLE 3                                                         ______________________________________                                        Ear thickness                                                                                              Mean decrease                                    Treatment     Number of ears treated                                                                       in oedema                                        ______________________________________                                        None          20             0                                                Topical Calaguala                                                                           20             32%                                              Pre-treatment Calaguala                                                                     20             32%                                              ______________________________________                                    

The thickness of the ears was measured after 2 hours. One Calagualagroup received the extract topically, 400 μg/ear, and another group hadbeen pre-treated for two weeks with about 1000 mg/kg/d Calaguala intheir drinking water. The significancies were p<0.001 for the localtreatment and p<0.005 for the oral treatment (Students t-test, onetailed).

Rat Paw Oedema

The rat paw oedema test is a classical way of expertimentallyinvestigating the effect of different compounds in acute inflammation.The most commonly used inducer is carrageenan which is injected to thehind paw of male Spraque Dawley rats. The mechanism of the oedemadevelopment which culminates after three hours is not completelyunderstood. In general, it is believed to involve stimulation ofprostaglandin synthesis, a fact that is supported by the inhibitoryactivity of cyclo-oxygenase inhibitors in this assay. More recent data,however, indicate that additional mechanisms are involved in the oedemadevelopment. For example, it has been shown that early phase of thecarrageenan induced rat paw oedema is partly dependent on PAF release(Caruso et al., Pharmacol. Res. 31(1), 67-72, 1995). PAF has also beenfound to potentiate the oedema induced by carrageenan and the known PAFreceptor antagonist, BN 52021, is capable of abolishing thisenhancement.

Several tests were performed with the methanolic Calaguala extract whichwas applied orally in doses 500-2000 mg/kg. As positive control thecyclo-oxygenase inhibitor Na-salicylate (200 mg/kg) was used. Arepresentative study is presented below:

                  TABLE 4                                                         ______________________________________                                        Effect of Calaguala methanolic extract on rat paw                             oedema induced by carrageenan.                                                                       Mean decrease                                          Treatment   No. of animals                                                                           in oedema  Significance                                ______________________________________                                        None        5          --         --                                          Na-salicylate                                                                             5          43%         p < 0.0005                                 Calaguala 500 mg/kg                                                                       5          19%        p < 0.05                                    Calaguala 1000 mg/kg                                                                      5          22%         p < 0.025                                  Calaguala 2000 mg/kg                                                                      5          21%        p < 0.05                                    ______________________________________                                    

The significancies were calculated using Students t-test (one tailed).

In another set of experiments rats were pre-treated for two weeks withCalaguala methanolic extract in their drinking water. No statisticallysignificant decrease in the oedema formation was observed when the ratswere challenged with carrangeenan on day 14. This finding is well inline with the theory of PAF being involved in the early phase of thecarrageenan induced oedema whereas the later phases would be aused bystimulation of prostaglandins.

The Calaguala extract has also been investigated for inhibitory activityon the enzyme cyclooxygenase using bovine seminal vessel microsomes(White et al., Prostaglandins 7, 123-9, 1974). Cyclooxygenase is thefirst enzyme in the reaction chain for conversion of arachidonic acid toprostaglandins. The extract failed to show any inhibitory activity inthis assay. This could be the explanation to why the inhibition causedby Calaguala in the carrageenen induced rat paw oedema does not increasewith increased concentrations of the extract: only a minor part of theoedema increase is caused by PAF and Calaguala is only capable ofinhibiting that part leaving the increase prostaglandin productionunaffected. Thus the concentration of 500 mg/kg is already sufficient tocause total inhibition of the PAF induced part.

Quantification of SQDG In Calaguala Methanolic Extract

Methanolic extract prepared from leaves of Polypodium decumanum has beenanalysed for its content of sulphonoglycolipid, SQDG.

5700 gram of finely ground plant material was extracted with methanolovernight, under stirring at room temperature. The process was repeatedthree times with a total of 35 liter methanol. The extract was filteredand dried and the yield was 337 gram, In order to get a detectablesignal for SQDG the methanolic extract was subjected to a preliminaryfractionation.

Initially the extract was dissolved in water and the lipids wereseparated from the polar components by partitioning to chloroform. Thechloroform extract was thereafter applied on a Sephadex LH20 columnwhich had been equilibrated with ethanol. The column was first elutedwith ethanol and thereafter with methanol. SQDG was selectively enrichedin the methanol fraction. The fractions eluting prior to the change ofeluent was examined by NMR for the presence of SQDG but only minortraces could be detected.

The HPLC analysis was carried out by using a straight phase system withpolyvinyl alcohol bonded silica column (T-column, 89° C., PVA-SIL,250×4.6 mm, 5μm), a flow rate of 1 ml/min, a light scattering detector(Sedex 45) and a mobile phase with a gradient system with hexane,iso-propanol, n-butanol, tetrahydrofuran, iso-octane, water and ammoniumacetate.

Commercially available SQDG was used as external standard and regressionanalysis was used to obtain an equation for a straight line. This wasthen used to calculate the amounts of SQDG present in the extract.

The amount SQDG present in the Calaguala extract was determined to be 4mg/g extract.

PAF Induced Exocytosis

In this test, which is also called the elastase assay, the effect of theextracts on inhibiting the exocytosis induced by PAF was examined. Thismodel was used to guide the fractionation of the extracts or isolatedcompounds.

Cell preparation: Peripheral blood anti-coagulated with heparin wasobtained from healthy volunteers at the University Hospital (in Uppsala,Sweden). After sedimentation with 10% Dextran T-500 (Pharmacia FineChemicals) for 30 min at 20° C. the supernatant was removed andcentrifuged at 200×g for 30 min. The sediments were treated with onevolume of ice-cold water for 21 s followed by the addition of 9 volumesof Mg²⁺ and Ca²⁺ free PBS in order to lyse the remaining erythrocytes.After centrifugation (200×g for 10 min at 4° C.) the leucocytes weresuspended in PBS (containing Mg²⁺ and Ca²⁺ and Cytochalasin B, Serva, 5μg/ml) at a concentration of 10-30×10⁶ cells/ml.

The assay was performed as described by Tuominen et al., Planta Medica58, 306-310 (1992). In short leucocytes were incubated at 37° C. in PBScontaining 2.5% BSA together with SAAVNA(N-succinyl-L-alanyl-L-alanyl-L-valine-p-nitroanilide, Bachem), theinhibitor in different concentrations or the vehicle (max. 0.1% DMSO)and PAF (Bachem) for 10 min. Blank tubes without PAF were run inparallel. The reaction was stopped by the addition of 2% citric acid andafter the centrifugation, the samples were measured in a UVspectrophotometer at 405 nm. The absorbance of the corresponding blanktube was subtracted from the sample and the inhibition of PAF inducedelastase release was calculated as the relative increase in absorbanceas compared to the vehicle. The samples were analyzed as 4-5concentrations ranging from 10⁻³ M to 10⁻⁷ M. They were dissolved in 10%DMSO (the isolated compound) or cyclodextrin (BN 52021, used as areference) and then diluted with the buffer. The final concentration ofDMSO or cyclodextrin never exceeded 0.1%.

The dose response curve of the SQDG of the formula III, and of the PAFreceptor antagonist ginkgolide BN 52021 is shown in FIG. 1. Each pointrepresents the mean of 2-4 experiments, all performed in duplicate. TheIC₅₀ value was determined to 10 μM for the compound of the invention andto 80 μM for BN 52021.

In another experiment, the activity of SQDG rich fractions of differentorigin, such as from Spirulina and from bladder-wrack, were compared tothe activity of SQDG from Calaguala, but no differences could beobserved.

In still another experiment different polar lipids were tested in themodel. Compounds such as MGDG and DGDG showed no inhibitory activity inconcentrations around the IC₅₀ of SQDG.

[³ H]PAF Binding Assay

The PAF induced exocytosis model described above is used for screeningfor PAF receptor antagonists since it is a rapid, reproducable andreliable so called functional assay for PAF. However, a number of otheractions, like elastase inhibition or membrane stabilisation, would alsoresult in an inhibitory effect in the model which consequently is notexclusive for PAF receptor antagonism. To elucidate if SQDG acts throughPAF receptor antagonism in the neutrophil, a receptor binding modelusing [3H]PAF as the radioligand was used.

Neutrophils are the most abundant circulating white blood cells and areusually the first cells to respond to an infectious or inflammatorystimulus. They are capable of phagocytosing appropriately opsonizedbacteria and may also upon stimulation with e.g. PAF, secretepotentially toxic oxygen metabolites and lysosomal enzymes, includingelastase, into the supernatant medium. These cells can also adhere tovascular endothelium and migrate towards the source of thechemoattractants. All these properties are a part of the response of theorganism to inflammatory or infectious disturbance.

Cell preparation: For the receptor binding experiments the cells wereprepared in the same way as described above except that concentratedsuspensions of human leukocytes in a CDP-adenine solution were used andin the final step the cells were suspended in the incubation buffer andthe cell count was adjusted to 3.1×10⁶ cells/ml.

[³ H]PAF (C₁₈ ; 1-O-[³ H]octadecyl-2-acetyl-sn-3-phosphocholine;Amersham, England) receptor binding was studied in humanpolymorphonuclear leukocytes (2.5×106 cells/ml) using a method recentlyreevaluated by us (Vasange et al., in press, 1996). Incubations wereperformed for 120 min at 20° C. in a total volume of 1.0 ml containing0.6 nM Na₂ HPO₄, 0.6 nM NaH₂ HPO₄ 25 mM Tris HCl, 130 mM NaCl, 5.5 mMKCl, 1.4 mM CaCl₂, 0.7 mM MgCl₂, 10 mM glucose, 0.5% BSA and 0.35-0.40nM [³ H]PAF. Final pH was 7.0 at the incubation temperature used.Non-specific binding was determined with 1 μM C16-PAF (Bachem,Switzerland). The incubation contents were rapidly filtered by vacuumover Whatman GF/C glass-fibre filters using a 12 well Skatron CellHarverster (Skatron A/S, Norway) and washed with 3×2 ml incubationbuffer containing 0.1% BSA. The filtration took less than 15 s. Filterswere also pre-soaked with incubation buffer containing 0.1% BSA.Following filtration, the filters were dried in an oven at 70° C. for 30min and equilibrated for two hours in the scintillation vials beforecounting in a Packard scintillator at an efficiency of 55%. Thecompetition binding assays were performed in triplicate in severalconcentrations (10⁻¹¹ -10⁻³ M) of the isolated compound and the receptorantagonists BN 50730 and BN 52021 as references. The compound and BN50730 were dissolved in 100% DMSO (10⁻² M), BN 52021 in cyclodextrin andthen further diluted with the incubation buffer.

The displacement curves of the ginkgolide BN 52021, thehetrapazine-derived PAF receptor antagonist BN 50730 and of the SQDGcompound of the formula III are presented in FIG. 2. The figure showsthe decrease on dpm measured as percentage of total bound ligand (Bt).The level of non-specific binding was determined to about 45% of the Bt.Each point represents the mean of 2-4 experiments, all performed intriplicate. The IC₅₀ value was determined to be 2 μM for the isolatedcompound, 25 μM for BN 52021 and 0.04 μM for BN 50730.

SUMMARY

Calaguala has in clinical trials been shown to have beneficial effectsin the treatment of psoriasis. One of the pathological findings in thepsoriatic skin is elevated amounts of the inflammatory mediator plateletactivating factor. The crude methanolic extract of Calaguala which gavethe effects in psoriasis also shows a dose dependent inhibition of thePAF induced elastase release in human neutrophils, which is a functionalassay for PAF receptor antagonism. When activity guided fractionation ofthe Calaguala extract was carried out, the main compound responsible forthe PAF activity was discovered to be SQDG. SQDG was also proven to actthrough binding to PAF receptors in human neutrophils indicating a truereceptor antagonistic property. Thus SQDG is believed to havetherapeutic potential in treatment of diseases, such as inflammatoryskin diseases. Another pathological finding in the psoriatic skin iselevated amounts of leukotriene B₄ which is known to be inhibited bypolyunsaturated fatty acids. The combination of unsaturated fatty acidsand SQDG, as in the Calaguala extract, should therefore be beneficial inthe treatment of skin inflammation.

What is claimed is:
 1. A method for the treatment or prophylaxis ofinflammatory skin diseases or disorders in a mammal in need of thereofcomprising administering to the said mammal a pharmaceutical compositioncomprising an SQDG having the formula ##STR8## wherein R¹ and R², whichare the same or different, are hydrogen or saturated or unsaturated,optionally hydroxy-substituted acyl groups, protonated or as a salt. 2.A method according to claim 1 wherein said SQDG has the formula ##STR9##wherein R¹ and R², which are the same or different, are hydrogen, acetylor acyl groups comprising 14-22 carbon atoms, preferably 16-18, andhaving up to 6 unsaturations, preferably 0-3.
 3. A method according toclaim 1 wherein said SQDG has the formula ##STR10## protonated or as asalt.
 4. A method according to claim 1 wherein said inflammatory skindisease or disorder is selected from the group consisting of atopicdermatitis, urticaria and psoriasis.
 5. A method according to claim 1wherein said inflammatory skin disease is a proliferative disease.
 6. Amethod according to claim 1 wherein said pharmaceutical compositionfurther comprises said SQDG in combination with a polyunsaturated fattyacid in the form of free fatty acids, monoacylglycerols, diacylglycerolsor triacylglycerols.
 7. A method according to claim 6 wherein saidinflammatory skin disease or disorder is psoriasis.
 8. A methodaccording to claim 6 wherein said polyunsaturated fatty acid is linoleicacid or linolenic acid.